Adriamycin Inhibits Human RH II/Gu RNA Helicase Activity by Binding to Its Substrate Kuichun Zhu, Dale Henning, Tomoo Iwakuma, Benigno C. Valdez, and Harris Busch 1 Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030 Received October 16, 1999 RNA helicases are enzymes important in RNA syn- thesis, processing, transport, and turnover. Human nucleolar RNA helicase II/Gu protein (RH II/Gu) was expressed in a baculovirus system. The purified re- combinant RH II/Gu protein has RNA helicase activity on a 5* tailed ds RNA substrate in vitro. We found that Adriamycin, a widely used anticancer drug, inhibited RH II/Gu helicase activity in a dose-dependent man- ner with an IC 50 of 40 mM. Adriamycin bound to the RNA substrate, and the binding was disrupted by boil- ing or treatment with 1% SDS, suggesting that the binding of Adriamycin to RNA is reversible. Adriamy- cin was also found by gel electrophoresis to bind to yeast tRNA to form slow-migrating complexes. These results suggest that Adriamycin can inhibit RNA syn- thesis or processing by binding to RNA substrates. © 1999 Academic Press RNA helicases catalyze the unwinding of double strand RNA to produce single strand RNA. The RNA helicase family represents a large family of proteins with conserved DEAD or DEAH sequences. Only a few of the family members have been demonstrated to have RNA helicase activity (1–2). RNA helicase activities are important in cellular processes such as translation initiation, ribosomal RNA synthesis, RNA splicing, RNA editing, RNA transport, and RNA turnover (1–3). Some RNA helicases, such as eukaryotic initiation fac- tor 4A and RNA helicase I, are capable of unwinding duplex RNA bidirectionally (4 –5). Some RNA heli- cases, such as human p68 protein and RNA helicase A, only unwind duplex RNA with 39 end overhangs (6 –7). Others, such as RNA helicase II/Gu protein (RH II/Gu), only unwind duplex RNA with 59 end overhangs (8 –9). RH II/Gu RNA helicase is a nucleolar protein with an apparent molecular mass of 100 kDa. It unwinds RNA duplex in a 59 to 39 direction in the presence of ATP and Mg 21 (8 –10). RH II/Gu RNA helicase has an RNA helicase domain near its N-terminal and an RNA fol- dase domain near its C-terminal (10). Interestingly, RH II/Gu protein was identified using an autoimmune serum from a patient with watermelon stomach dis- ease (9). The physiological function of RH II/Gu RNA helicase has not been fully investigated, but prelimi- nary studies suggest that it may be involved in ribo- somal RNA synthesis due to its unwinding and folding activities (10 –11). Despite the wide use of Adriamycin in cancer chemo- therapy for decades, the mechanisms of the anticancer and cytotoxic effects of the drug are not completely understood (12). This study shows that Adriamycin inhibits RH II/Gu RNA helicase activity in a dose- dependent manner by binding to its RNA substrate, and that Adriamycin retards the mobility of synthetic RNA substrates as well as yeast tRNA. MATERIALS AND METHODS Expression and purification of RH II/Gu RNA helicase protein. RH II/Gu RNA helicase cDNA was cloned as described previously (9). The full length cDNA was cloned in frame into a baculovirus transfer vector pAcHLT-B (Pharmingen, San Diego, CA) at the Nco I/Pst I sites so that a His tag is upstream of the cloning site. The resulting clone was confirmed by its DNA sequence. Transfection of Sf 9 insect cells and amplification of recombinant virus were performed accord- ing to the manufacturer’s protocol. Cells were harvested 96 hours post-infection. The cell pellets were lysed in 10 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1% Triton X-100, 10 mM sodium fluoride, 10 mM sodium phosphate, 10 mM sodium pyrophosphate, 1 mM PMSF, 1.5 mM aprotinin, 20 mM leupeptin, 14 mM pepstatin A, 10 mg/ml of phenanthroline, and 16 mg/ml of benzamidine. Lysed cells were cen- trifuged at 12,000 3 g for 20 min at 4°C. The pellet was extracted with the same lysis buffer containing 800 mM NaCl. After centrifu- gation, the supernatant was loaded onto a Talon metal affinity col- umn for His tagged proteins (Clontech, Palo Alto, CA). The loaded column was washed sequentially with the lysis buffer (20 volumes), lysis buffer containing 10 mM imidazole (5 volumes), and lysis buffer containing 20 mM imidazole (5 volumes). Recombinant protein was eluted with the lysis buffer containing 100 mM imidazole. The eluted protein was pooled and dialyzed against 20 mM Hepes buffer, pH 7.6, 1 mM DTT, 0.1 mM EDTA, 100 mM KCl, and 10% glycerol. Protein concentrations were determined with a Bio-Rad protein assay kit (Hercules, CA) using BSA as the standard. A fraction of purified protein was analyzed on SDS-PAGE and stained with a Bio-Rad silver plus kit according to the manufacturer’s protocol. 1 To whom all correspondence should be addressed. Fax 713-798- 3145. E-mail: hbusch@bcm.tmc.edu. Biochemical and Biophysical Research Communications 266, 361–365 (1999) Article ID bbrc.1999.1815, available online at http://www.idealibrary.com on 361 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.